Betulin and its derivatives – precursors of new drugs

Available online at www.worldscientificnews.com

( Received 26 March 2019; Accepted 12 April 2019; Date of Publication 13 April 2019 )

WSN 127(3) (2019) 123-138 EISSN 2392-2192

Betulin and its derivatives – precursors of new drugs

Małgorzata Drąg-Zalesińska*, Sylwia Borska

Division of Histology and Embryology, Department of Human Morphology and Embryology, Faculty of Medicine, Wroclaw Medical University, 6a Chalubinskiego Str., 50-368 Wroclaw, Poland

*E-mail address: [email protected]

ABSTRACT

White birch bark extract, used in traditional medicine, has many medical properties. Its main

components are betulin and betulinic acid - lupane type pentacyclic triterpenes. The discovery of the

anti-tumor and anti-viral properties of betulinic acid has become an impulse to modify the structures of

these compounds in order to obtain better activity. Over twenty years of research, several derivatives

have been selected that have significantly better anti-tumor or antiviral properties than their precursors.

This paper presents all new derivatives of betulin and betulinic acid, which have been tested in phase I,

II or III of clinical trials. Clinical experiments on animals with natural cancers have also been described.

The results obtained by various research teams give hope for the potential use of these compounds in

therapy. At the same time, the presented analysis shows that the implementation of the new drug on the

market is a process that lasts 10-15 years, which, despite the involvement of huge financial and human

resources, does not guarantee success.

Keywords: betulin, betulinic acid, bevirimat, oleogel-S10, episalvan

1. INTRODUCTION

The search for new medicinal substances is an extremely complex, expensive and time-

consuming process. Implementation of the new drug on the market takes about 10-15 years,

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World Scientific News 127(3) (2019) 123-138

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and the total cost of such a venture is several billion dollars. Despite such large financial

expenditures and the involvement of many specialists, there is no guarantee of success. Many

promising substances are eliminated after many years of laboratory testing at the stage of

clinical trials, because they appear to cause serious side effects [1].

Modern methods of searching for new drugs are based on in silico technologies. The first

step in designing a drug is to determine the biological target, for example a protein whose

function is to be modulated by the designed molecule. Then structure of the so-called lead

structure is determined. The next steps are the optimisation of pharmacokinetic and

pharmacodynamic properties as well as confirmation of the activity and mechanism of action

of the new substance in in vitro studies on cellular and in vivo models in experimental animals.

Pre-clinical and clinical trials are the last stage in the study of new drugs. Currently, the

pharmaceutical industry uses advanced in silico technologies including molecular modeling,

bioinformatic techniques - genomics, proteomics and chemoinformatics - combinatorial

libraries, QSAR (Quantitative Structure-Activity Relationship), QSPR (Quantitative Structure-

Property Relationship) and prediction properties of ADMET (Absorption, Distribution,

Metabolism, Excretion, Toxicity) [1, 2].

In spite of the development of in silico technology, the search for new natural substances

with biological activity, of which the plant kingdom is a virtually unlimited source, is still valid.

For a long time scientists have been interested in the so-called secondary metabolites, especially

those found in plants used in traditional medicine. For many years, the function of secondary

metabolites was unknown, and therefore they were classified as metabolic waste. Recently,

however, it has been shown that they play an important role in the adaptation of the plant to the

environment through interaction with other organisms - pathogens or competitors. One of the

most numerous secondary metabolite groups are terpenes and terpenoids [3, 4].

The terpene biosynthesis takes place by combining five-carbon isoprene units (C5H8) into

chain or ring forms to form a compound of general formula (C5H8)n. Derivatives of terpenes,

which contain, in addition to the hydrocarbon skeleton, any heteroatoms are called terpenoids.

Depending on the size of the molecule, the following are distinguished: hemiterpenes (n = 1),

monoterpenes (n = 2), sesquiterpenes (n = 3), diterpenes (n = 4), triterpenes (n = 6),

tetraterpenes (n = 8) and polyterpenes. Commonly known terpenes are isovalerate acid

(hemiterpen), geraniol, menthol, camphor (monoterpenes), lycopene and carotenes

(tetraterpenes) and natural gum (polyterpene). An example of a linear triterpene is squalene, an

oil component derived from shark liver.

Pentacyclic triterpenes are betulin and betulinic acid. [5]

2. PENTACYKLIC TRITERPENES

The pentacyclic triterpene skeleton is composed of six isoprene units and is formed by

cyclisation of the squalene molecule. The compounds are characterised by the presence of five

six-carbon or four six-carbon rings and one five-carbon ring [5]. Depending on the structure,

terpenes are divided into 5 types, which include various derivatives:

1) lupane type: lupeol, betulin, and betulinic acid;

2) oleanane type: oleanolic acid, ß-amyrin, erythrodiol;

3) ursane type: ursolic acid, α-amyrin, uvaol;

4) taraxastane type: taraxasterol;


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5) taraxerane type: taraxerol. [5, 6].

The general scheme for the structure of lupane type pentacyclic triterpenes is shown in

Fig. 1.

Figure 1. The general chemical structure of the lupane type pentacyclic triterpenes.

R = OH – betulin; B); R = COOH – betulinic acid; C) R = CH3 – lupeol.

Triterpene compounds are present in the bark, resin, fruit peel, leaves, flowers, essential

oils and seeds, performing a protective function against microorganisms and insects. Their

occurrence in the plant kingdom is common, and the same derivatives of lupane have been

found in more than 300 species of plants, among others Betula alba, Olea europeae,

Rosmarinus officinalis, Sambucus nigra, Platanus acerifolia, Vitis vinifera, Calendula

officinalis, Salvia officinalis [6].

Plants with a high content of triterpenes are used in phytotherapy due to their healing

properties. Usually, these are well-known plants used in folk medicine with anti-inflammatory,

anti-bacterial, anti-fungal, anti-cancer and anti-allergic properties [6, 7].

Triterpene compounds are the subject of numerous phytochemical and pharmacological

studies, and the increase in interest in betulinic acid and betulin dates from the publication of

Pisha et al. in Nature Medicine in 1995 [8].

Pisha et al. were the first to report that betulinic acid is cytotoxic to human melanoma

line MEL-1, -2, -3, -4 with no toxic effects on normal cells. The experiments were performed

in vitro on cell lines and in vivo on mice. This publication was cited almost 600 times over 24

years.


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3. BETULIN AND BETULINIC ACID

Betulin (BE) is one of the most commonly found in nature triterpenes. The richest source

is the bark of white birch species (Betula sp.): B.verrucosa, son B. pendula, B. pubescens, B.

alba, in which it occurs in the amount of 20-35% [9]. BE occurs in the form of crystal clusters

in large, thin-walled cells, arising in the birch bark in the spring. It is betulin that gives the bark

of these trees a white colour [9]. This compound was isolated for the first time from birch bark

and described by Lowitz in 1788. The isolation of BE from birch bark can be carried out by

sublimation or by extraction with organic solvents such as chloroform, acetone, ethanol,

tetrahydrofuran, n-heptane. The content of BE in the bark extracts of various birch species is

70-80%, the rest is made up of other terpenes: betulinic acid (4-12%), lupeol, betulinaldehyde,

oleanolic acid. The exact structure of betulin was established in 1952 [10, 11]. This compound

is made of a thirty-carbon skeleton, made up of four six-membered rings and one five-

membered, trans-connected in relation to each other. This structure reminds steroids in

structure. The chemical activity of betulin depends on the presence of hydroxyl groups at C3

and C28 and the isopropenyl group at C19 [10].

The natural derivative of betulin is betulinic acid (BA), which differs from BE by the

presence of a carboxylic group instead of hydroxymethyl at C17 (Fig.1). BA occurs in the bark

of birches and other plant species, but in an amount that does not allow for efficient extraction,

that is why BE is a substrate for the synthesis of this natural derivative. It should be emphasised

that both compounds are characterised by very low solubility in water. The chemical synthesis

of BA relies on the oxidation of BE with Jones reagent (Na2Cr2O7, H2SO4 in acetone, at a

temperature close to 0 °C), and then reduction of the obtained betulonic acid with NaBH4 in

tetrahydrofuran. The specific betulinic acid epimer is isolated by crystallisation. This method

is unfortunately not very efficient (about 60%). A more efficient reaction (86%) is

chemoselective oxidation of the primary hydroxyl group of betulin directly to the carboxylic

ones [12].

So far, total synthesis of BE and BA has not been carried out in laboratory conditions.

The largest, constantly renewable source of betulin is the birch bark. German company Birken

Gmbh. has developed a new highly efficient way of obtaining betulin from a birch cork

cambium (white part of the birch bark) which is a waste material from pulp mills. In the patent-

protected method (EP 1 758 555 B1) betulin of 80-90% purity is obtained [13].

BE and BA are excellent substrates, or structures leading to further chemical

transformations and the creation of new derivatives with stronger anti-cancer, anti-viral or anti-

microbial properties. The latest report by Sousa et al. from 2019 discusses in detail the

modifications of BE and BA, which were published in 2013-2018 [14]. The study presents over

200 new derivatives, including the synthesis of betulin esters with L-amino acids, which was

carried out in the years 2013-2015 by our research team [15]. The possibilities of modifying

BE and BA by simple transformations are described: amination (amide and amine derivatives),

esterification, sulfonation, alkylation.

The creation of new compounds by click chemistry and modifications by hydroxylation

or aldol condensation are discussed. The published report shows that modifications of the

betulin and betulinic acid molecules mainly concern active groups at C3 and/or C28. New

derivatives are designed to increase anti-tumor, anti-HIV and anti-malarial properties and are

usually characterised by much better solubility in water than BE and BA [14, 15].


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3. 1. Anti-tumor activity of betulinic acid and its derivatives

The first report on the anti-tumor activity of betulinic acid was Pisha's publication in

1995, in which selective cytotoxicity of this compound was found relative to human melanoma

cells MEL-1, -2, -3, -4. In vivo studies - no BA toxicity for experimental animals - indicated the

potential of BA in human melanoma therapy [8]. In 2001, Salti et al. reported the protective

effect of BA against DNA damage induced by UV-C (254 nm) in cultured congenital

melanocyte cells (primary lines were derived from a biopsy of a congenital melanocytic nevi

from a healthy patient) [16].

Currently, the mechanism of anti-tumor activity of BA is well known and comes down to

three main points:

1) anti-proliferative activity by inhibiting topoisomerase I and II in tumor cells;

2) activation of the mitochondrial apoptosis pathway in cancer cells;

3) the ability to inhibit angiogenesis within the tumor.

BA is an inhibitor of eukaryotic topoisomerase I, it causes its inactivation and prevents

the formation of DNA-replicating enzyme complexes. This prevents replication and inhibits the

proliferation of cancer cells. In turn, the inhibition of topoisomerase II leads to the inhibition of

DNA repair processes [17].

The most important anti-tumor mechanism of action of BA is its ability to induce the

mitochondrial pathway of apoptosis. Anti-cancer therapy is based mainly on the initiation of

the mitochondrial pathway of apoptosis [18-21]. Many chemotherapeutic agents and

radiotherapy have such an effect. BA induces the formation of free radicals (ROS) in various

tumor lines, what increases the permeability of the outer mitochondrial membrane.

The regulation of the mitochondrial membrane permeability depends on the expression

of proapoptotic proteins, e.g. Bax, Bak, Bad and on the expression of antiapoptotic proteins,

e.g. Bcl-2, Bcl-XL, and Mcl-1.

BA has the ability to modulate the level of expression of these proteins, what may increase

the level of mitochondrial membrane permeability and induce the process of apoptosis in cancer

cells. Increasing the permeability of mitochondrial membranes results in the release into the

cytosol of apoptogenic proteins from the intermembrane space of mitochondria: cytochrome c

and AIF (Apoptosis Induction Factor).

Cytochrome c activates caspase-8, and it activates caspase-3, what in turn leads to the

activation of endonucleases. The next step is the fragmentation of nuclear DNA, cell shrinkage

and its phagocytosis by cells of the immune system.

Inhibition of angiogenesis is the result of inhibiting by BA the aminopeptidase N activity

- an enzyme of key importance for the angiogenesis process [19]. BA also affects the selective,

proteasome-dependent degradation of transcription factors: Sp1, Sp3 and Sp4, which regulate

the expression of vascular endothelial growth factor (VEGF) [20, 22].

The study by Ali-Sayed et al. summarises many years of research on the anti-cancer

activity of BA. BA exhibits anticancer activity against a variety of cell lines, e.g. melanoma,

neuroblastoma, medulloblastoma, glioblastoma multiforme, ovarian, colon, prostate, lung,

breast, cervix cancer. It should be emphasised that BA is characterised by selective cytotoxicity

at low concentrations in relation to tumor cells, with the lack of toxicity of the same doses in

relation to normal cells. There was no BA toxicity in relation to human normal fibroblasts,

lymphocytes, keratinocytes [15, 18, 23].


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In the years 2006-2013, a phase I/II clinical trial (NCT00346502, University of Illinois,

Chicago, USA, www.clinicaltrials.gov) was carried out to evaluate the safety and effectiveness

of experimental ointment with 20% BA for the treatment of dysplastic melanocytic nevus. It

was planned to include 28 patients from 200 after the initial screening in the study. The study

consisted of using ointment with 20% BA for dysplastic nevus for four weeks. After this period,

the dysplastic nevus treated was removed surgically. For control, a similar nevus not treated

with an ointment was removed.

The removed nevi were examined histopathologically. Unfortunately, the results of the

experiments were not published. The study was suspended in 2013 due to lack of financing.

Simone Fulda published the information on this program for the first time in 2008 without

providing any references [19]. Probably the researcher collaborated with the American centre

in the field of research on the mechanism of anti-tumor activity of BA. Although the clinical

trial was completed in 2013, there are still publications which emphasise that BA is currently

in the phase I/II of clinical trials assessing its effectiveness in the treatment of dysplastic nevus

[18, 24].

The next phase I pilot clinical trial (NCT00701987, www.clinicalstrials.gov) to evaluate

the safety, tolerability and effectiveness of ALS-357 (BA) in patients with cutaneous metastatic

melanoma was conducted in 2008-2009. 12 participants took part in the study. The drug was

applied topically in the form of an ointment for 4 weeks in four arms: twice a week, every other

day, once a day and twice a day. Unfortunately, the BA concentration in the preparation was

not included in the study information. In biopsies, apoptosis was evaluated by the TUNEL

method and the expression of genes and proteins of the apoptotic pathway (day: 15, 29, 43). At

each visit, the ALS-357 concentration in serum (day: 1, 8, 15, 22, 29, 43) was evaluated.

Unfortunately, no research results have been published.

Clinical studies evaluating the effectiveness of BA in melanoma treatment may have been

discontinued due to the progress in the treatment of this cancer, which took place in 2011, when

FDA (Food and Drug Administration, USA) registered a new drug, an inhibitor BRAF,

vemurafenib for the treatment of melanoma [25]. This initiated a new era in the treatment of

melanoma. Betulinic acid derivatives that show significantly greater cytotoxicity to tumor cells

than BA alone are NVX-207 and B10. The molecular structures are shown in Fig. 2.

Figure 2. Chemical structure of betulinic acid (BA), NVX-207 and B10.

The first report on the significant anti-cancer activity of NVX-2017 in in vitro and in vivo

studies dates from 2009 [26]. The activity of the compound (mean IC50 = 3.5 μM) in vitro was


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determined using normal and tumor cell lines (human and canine). The studies confirmed that

NVX-207 activates the mitochondrial pathway of apoptosis. Phase I/II trials in dogs with

natural tumors showed excellent clinical response after the topical treatment with NVX-207

(application once per week). Complete tumor remission was obtained in all five treated animals

[27]. In 2016, Liebscher et al. presented the results of research conducted on melanoma tumor

lines (MelDuWi and MellJess) and human melanoma lines (A375) using BA and its derivatives:

NVX-207 and B10 [28].

It has been shown that all three compounds, in particular NVX-207, are characterised by

high cytotoxicity towards equine melanoma cells, activating the mitochondrial apoptosis

pathway. The studies were also carried out in vivo on 2 horses (13- and 18-year-old) suffering

from melanoma. The experiment consisted of intratumoral injections of NVX-207 once a week

for the next 19 weeks. No irritation was observed, the animals tolerated the therapy well. In

laboratory tests, no abnormalities were observed, except for transient increases in hepatic

transaminases [28]. One horse was euthanised in the 19th week of the experiment due to colon

torsion. Unfortunately, it has not been described whether changes in melanoma lesions in the

form of tumor regression or cure were observed.

Compound B10, a glycosylated derivative of BA, has a different mechanism of cytotoxic

activity than BA and NVX-2017. It turned out that B10 induces autophagy, destabilises

lysosomes and releases cathepsins Z and B to the cytoplasm [29]. This report and studies from

2014 indicate the possibility of using B10 in the treatment of tumors under hypoxic conditions

due to the increased cytotoxicity of this compound in hypoxia, e.g. in the treatment of malignant

glioma [30].

Despite the large potential anti-tumor activity and the well-known mechanism of

cytotoxicity against cancer cells, BA and its derivatives have not been used in therapy so far.

In addition to in vitro experimental studies, there are reports of the use of two potential drug

precursors in cancer therapy in dogs and equine melanoma.

3. 2. Anti-viral activity of betulinic acid and its derivatives

The search for herbal medicinal agents capable to fight HIV infection resulted in the

discovery in 1994 of the anti-viral properties of the extract from the leaves of Syzygium

claviflorum. The active compounds of this extract are betulinic acid and its derivative of

platanic acid [31]. In the course of further research, a semi-synthetic derivative of BA with

stronger anti-viral properties was discovered.

This derivative, being 3-O-(3’,3’-dimethylsuccinyl) betulinic acid, is now known as

bevirimat. The structure of the molecule is shown in Fig. 3.

This preparation has very strong inhibitory properties of HIV-1 replication, because as a

protease inhibitor it leads to inhibition of virus maturation in infected lymphocytes. This

compound has been characterised as a maturation inhibitor - a representative of a new group of

anti-HIV drugs [32]. In 2007, two reports were published describing the results of the first and

the second phase of bevirimat clinical trials [33, 34].

The compound was evaluated for safety, pharmacokinetics and pharmacodynamics in a

double-blind study in healthy volunteers and in HIV-infected adults (phase IIa and IIb, 57

people infected with HIV). The drug has been found to be safe and well tolerated, with no dose-

limiting toxicity or serious side effects. The most frequently reported adverse effects of

bevirimat were headache and discomfort in the throat, but none of the study participants

discontinued using the preparation [33, 34].


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In the years 2008-2009 another phase II of clinical trial was conducted (NCT01097070,

www.clinicaltrials.gov), which included 35 HIV-positive volunteers. Then, clinical trials of the

drug were suspended due to the take over of rights to bevirimat by the company Myriad

Genetics, which stopped work on HIV maturation inhibitors in 2010.

Figure 3. Chemical structure of bevirimat [3-O-(3', 3'- dimethylsuccinyl) betulinic acid];

BA – betulinic acid

Further in vitro studies of bevirimat showed a build-up of drug resistance, what was

related to nucleotide polymorphisms at the CD capsid/SP1 protein cleavage site and mutations

at the CD/SP1 site. These mutations were not found during clinical trials, what may indicate

their much slower appearance in in vivo conditions [35, 36]. Therefore, clinical studies of

bevirimat were definitively terminated. However, in vitro experimental studies were continued,

what led to the discovery of second generation maturation inhibitors, the representative of

which is BMS-9551766. The chemical structure of the molecule is shown in Figure 4.

The BMS-9551766 synthesis pathway (GSK3532795) was published in 2016 [37]. Phase

II of clinical trials sponsored by ViiV Healthcare were conducted in cooperation with

GlaxoSmithKline in 2013-2014 (NCT01803074, www.clinicaltrials.gov) and in 2015-2016

(NCT02415595, www.clinicaltrials.gov). Unfortunately, they were discontinued due to the

strong gastrointestinal intolerance of the preparation and the increase in drug resistance in

patients [38].

The representative of the second generation inhibitors is also a derivative GSK2838232.

The chemical structure of the molecule is shown in Fig. 5. This compound in 2013-2016 was

tested in phase I clinical trials in four different programs (NCT01802918, NCT02289482,

NCT02289495, NCT02795754, www.clinicaltrials.gov) sponsored by GlaxoSmithKline. The

results of these studies were published in 2018 [39]. A good safety profile as well as


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pharmacokinetic and dynamic properties were found to allow the continuation of clinical trials

in phase II in 2017-2018 (NCT03045861, www.clinicaltrials.gov). It has been given to HIV-

infected patients in combination with other antiretroviral therapy: Cobicistat. The results of

these studies have not yet been published.

Figure 4. Chemical structure of BMS-9551766 and BA – betulinic acid.

Figure 5. Chemical structure of GSK-2838232 and BA – betulinic acid.

Studies on the anti-viral activity of betulinic acid contributed to the discovery of a new

group of anti-HIV drugs – first generation maturation inhibitors, which is represented by

bevirimat and second generation maturation inhibitors - BMS-9551766 (GSK3532795) and

GSK2838232. Although none of these molecules have been introduced for the treatment of


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HIV, these studies have had a significant impact on the progress in HIV treatment that has

occurred over the past 20 years.

3. 3. Anti-tumor activity of betulin

Betulin (BE) has much lower anti-tumor activity than BA. It inhibits weaker the

proliferation of melanoma cells in comparison with BA and its derivatives. It has been shown

that betaulin induces the mitochondrial pathway of apoptosis in lung cancer, breast cancer,

colorectal cancer, and cervical cancer [11]. However, reports of BE anti-tumor activity are few

and have not been a starting point for further studies of BE anti-tumor activity. During the last

20 years, the German company Birken Gmbh conducted research on the biological activity of

birch bark extract (TE - triterpen extract). A first description of an emulsion containing TE is

provided in the international patent EE 200200552(A) (PL 202948) [13].

The TE used to prepare the emulsion contains at least 80% betulin, a maximum of 10%

betulinic acid, a maximum of 3% lupeol and 4% oleanolic acid. The emulsion, in turn, contains

from 2-10% of TE and avocado oil, almond oil, water, glycerol or urea. Due to its preservative

(anti-bacterial, anti-fungal) and emulsifying properties of triterpenes, this composition does not

have to contain additional preservatives. The emulsion was used for the preparation of

medicaments in the form of ointment, cream, gel for application to the skin for the treatment

of, among others, precancerous conditions and damaged skin. The patent also describes in detail

the toxicological tests carried out on experimental animals - mice and rats. During

intraperitoneal administration of TE, toxic symptoms were seen in mice and rats at doses above

500 mg/kg body weight. It was also found that one-off subcutaneous administration of the

product at 2000 mg/kg body weight over 14 days does not cause mortality. Another study on

TE kinetics was performed on rats and dogs by subcutaneous and intraperitoneal administration.

The experiment showed no toxicity of TE for rats and dogs at a daily dose of 300 mg/kg body

weight during a 28-day application [40].

The first report on the effectiveness of TE in the treatment of the precancerous state of

the skin - AK (actinic keratosis) comes from 2006 [41]. 28 patients with AK were involved in

this pilot study, 14 patients were treated with TE only, and 14 patients underwent TE

cryotherapy and ointment with TE. The results showed the effectiveness of ointment with TE

in the treatment of AK with very good tolerability, without side effects. In the next phase II

clinical trial, conducted on a group of 45 patients who received oleogel with TE, the possibility

of using this preparation as an effective medicine for AK treatment was confirmed [42].

In this study, an improved formula of the original formulation was used, which was

named Oleogel-S10. This formula has patent protection in Europe since 2004 and in the US

since 2010 [43, 44].

In the years 2008-2010, a multi-centre, randomised, double-blind phase II trial was

conducted to evaluate the effectiveness of Oleogel-S10 in the treatment of AK and to plan phase

III trials (NCT00786994, www.clinicaltrials.gov). Unfortunately, the results published in 2015

did not confirm the effectiveness of Oleogel-S10 in the treatment of AK on a group of 165

participants. It was only shown that the drug was well tolerated during the 3-month period of

administration [45].

Unfortunately, the search for an effective preparation in the treatment of the precancerous

skin condition has not been successful. However, it turned out that Oleogel-S10 can be used in

other skin diseases, what is associated with anti-inflammatory, anti-microbial and wound

healing properties in the case of birch bark extract.


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3. 4. Anti-inflammatory, anti-microbial and wound healing effects of pentacyclic

triterpenes

Already in 1899, Wheeler stated that betulin had antiseptic properties and he used it to

sterilise dressing materials and wound patches [13]. There are reports of anti-bacterial and anti-

fungal effects of betulin and its derivatives on the bacterial strains of Enterobacter aerogenes,

Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus and

on fungi strain Candida albicans. Anti-bacterial properties are also demonstrated by lupeol,

oleanolic acid and betulin acid, which are components of the birch bark extract [12, 43].

Triterpenes are known to have anti-inflammatory activity by inhibiting enzymes involved

in the inflammatory reaction such as: phospholipase A2, cyclooxygenase, nitric oxide synthase.

In addition, they reduce the production of prostaglandins and proinflammatory cytokines: tumor

necrosis factor (TNF-α), interferon (INF-γ) and interleukins. These properties are characteristic

in particular for betulin, betulinic acid, lupeol and oleanolic acid [7, 46, 47].

The Oleogel-S10 formula is slightly different from the original emulsion developed at

Birken Gmbh. Instead of avocado and almond oil, sunflower oil was used, with the same

composition of triterpenes [45]. The product has successfully passed phase III of clinical trials

(NCT01657305, NCT01807650, www.clinicaltrials.gov). Three phase III trials were conducted

to evaluate the effectiveness and safety of this product. Two studies concerned wounds from

the sites of skin fragment removal for split-thickness skin graft (STSG) (219 patients) [48, 49,

50]. One study was conducted in patients with second degree burns (61 people) [51]. In all

studies, the acceleration of epithelization of wounds and the creation of a more elastic scar after

the use of the tested drug was demonstrated.

The preparation affects all three phases of wound healing: inflammation, migration and

differentiation. After activation of the inflammatory phase, faster epithelization is connected

with the migration and differentiation of keratinocytes [52, 53]. In 2016, Oleogel-S10 was

registered by EMA (European Medicines Agency, EU) as a preparation for treating second-

degree burns with the name Episalvan. The drug is available on prescription. In 2018, Episalvan

also obtained a positive FDA opinion and is currently in phase III of clinical trials

(NCT03068780, www.clinicastrials.gov) to evaluate the efficacy and safety of use in the

treatment of difficult to heal wounds in Epidermolysis bullosa (EB). EB is a rare group of

genetically conditioned skin diseases characterised by the formation of blisters on the skin after

even minor injuries. There is currently no effective treatment for EB. The first reports from

phase II trials show faster re-epithelization of EB wounds following the use of Episalvan [54].

A summary of the results of phase III clinical trials is planned for 2020 [55]. Oleogel-S10

(Episalvan) is the first registered medicine containing a herbal ingredient - an active triterpene

extract, mainly composed of betulin, derived from birch bark. Undoubtedly, this is a new and

significant achievement in phytotherapy.

4. CONCLUSIONS

Betulin and betulin acid due to their chemical structure are excellent structures leading to

the formation of new derivatives with potentially better anti-tumor, anti-viral or anti-

inflammatory properties. Undoubtedly, the success of studies on the anti-HIV effect of betulinic

acid was the discovery of a new group of antiretroviral drugs - maturation inhibitors. In turn,


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many years of Birken Gmbh's experiments contributed to the marketing of the first medicine

containing a triterpene extract intended for wound healing. There are still experimental and

clinical trials in numerous centres, and there will certainly be new reports on the use of lupane

type pentacyclic triterpenes in therapy.

ACKNOWLEDGEMENTS

The authors give thanks to Professor Maciej Zabel on valuable comments to the manuscript and Michał Kulus for

preparing the figures for this study.

References

[1] Bielska E, Lucas X, Czerwoniec A, Kasprzak JM, Kaminska KH, Bujnicki JM. Virtul

screening strategies in drug design – methods and applications. Journal of

Biotechnology, Computational Biology and Bionanotechnology 2011; 92: 249-264

[2] Klein S. The use of biorelevant dissolution media to forecast the in vivo performance of

a drug. AAPS J 2010; 12: 397-406

[3] Dinda B, Dednath S, Mohanta B, Harigaya Y. Naturally occurring triterpenoid

saponins. Chem Biodivers 2010; 10: 2327-2580

[4] Sumner LW, Mendes P, Dixon RA. Plant metabolomics: large-scale phytochemistry in

the functional genomics era. Phytochemistry. 2003; 62: 817-836

[5] Xu R, Fazio GC, Matsuda SPT. On the origins of triterpenoid skeletal diversity.

Phytochemistry 2003; 65: 261-291

[6] Jäger S, Trojan H, Kopp T, Laszczyk MN, Scheffler A. Pentacyclic triterpene

distribution in various plants - rich sources for a new group of multi-potent plant

extracts. Molecules 2009; 4: 2016-2031

[7] Rastogi S, Pandey MM, Kumar Singh Rawat A. Medicinal plants of the genus Betula-

traditional uses and photochemical-pharmacological review. J Ethnopharmacol 2015;

159: 62-83

[8] Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, Beecher CW,

Fong HH, Kinghorn AD, Brown DM, et al. Discovery of betulinic acid as a selective

inhibitor of human melanoma that functions by induction of apoptosis. Nat Med 1995;

1: 1046-1051

[9] Krasutsky PA. Birch barkresearch and development. Nat Prod Rep 2006; 23: 919-942

[10] Alakurtti S, Mäkelä T, Koskimies S, Yli-Kauhaluoma J. Pharmacological properties of

the ubiquitous natural product betulin. Eur J Pharm Sci. 2006; 29: 1-13

[11] Król SK, Kiełbus M, Rivero-Muller A, Stepulak A. Comprehensive review on betulin

as a potent aticancer agent. BioMed Research International Volume 2015, Article ID

584189, 11 pages. http://dx.doi.org/10.1155/2015/584189


-135-

[12] Csuk R, Schuck K, Schafer R. A practical synthesis of betulinic acid. Tetrahed Lett

2006; 47: 8769-8770

[13] Scheffler A. Emulsion containing a plant extract, method for producing said emulsion

and for obtaining a plant extract. European Patent Office. Patent No. EE 200200552(A)

2004.

[14] Sousa JLC, Freire CSR, Silvestre AJD, Silva AMS. Recent Developments in the

Functionalization of Betulinic Acid and Its Natural Analogues: A Route to New

Bioactive Compounds. Molecules 2019; 24: E355

[15] Drąg-Zalesinska M, Wysocka T, Borska S, Drąg M, Poręba M, Choromanska A,

Kulbacka J, Saczko J. The new esters derivatives of betulin and betulinic acid in

epidermoid squamous carcinoma treatment - In vitro studies. Biomed Pharmacother

2015; 72: 91-97.

[16] Salti GI, Kichina JV, Das Gupta TK, Uddin S, Bratescu L, Pezzuto JM, Mehta RG,

Constantinou AI. Betulinic acid reduces ultraviolet -C-induced DNA breakage in

congenital melanocytic naeval cells: evidence for a potential role as a chemopreventive

agent. Melanoma Res 2001; 11: 99-104

[17] Dillon LW, Pierce LC, Lehman CE, Nikiforov YE, Wang YH. DNA topoisomerases

participate in fragility of the oncogene RET. PLoS One 2013; 11: e75741

[18] Ali-Seyed M, Jantan I, Vijayaraghavan K, Bukhari SN. Betulinic Acid: Recent

Advances in Chemical Modifications, Effective Delivery, and Molecular Mechanisms

of a Promising Anticancer Therapy. Chem Biol Drug Des 2016; 87: 517-536

[19] Fulda S. Betulinic Acid for cancer treatment and prevention. Int J Mol Sci 2008; 9:

1096-1107

[20] Laszczyk MN. Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools

in cancer therapy. Planta Med. 2009; 75: 1549-1560

[21] Tan Y, Yu R, Pezzuto JM. Betulinic acid-induced programmed cell death in human

melanoma cells involves mitogen-activated protein kinase activation. Clin Cancer Res

2003; 9: 2866-2875

[22] Chintharlapalli S, Papineni S, Ramaiah SK, Safe S. Betulinic acid inhibits prostate

cancer growth through inhibition of specificity protein transcription factors. Cancer Res

2007; 67: 2816-2823

[23] Drąg-Zalesinska M, Drąg M, Poręba M, Borska S, Kulbacka J, Saczko J. Anticancer

properties of ester derivatives of betulin in human metastatic melanoma cells (Me-45).

Cancer Cell Int 2017; 17: 4

[24] Csuk R. Betulinic acid and its derivatives: a patent review (2008-2013) Expert Opin

Ther Pat 2014; 24: 913-923

[25] Miller FG, Joffe S. Equipoise and the dilemma of randomized clinical trials. N Engl J

Med 2011; 3: 476-480


-136-

[26] Willmann M, Wacheck V, Buckley J, Nagy K, Thalhammer J, Paschke R, Triche T,

Jansen B, Selzer E. Characterization of NVX-207, a novel betulinic acid-derived anti-

cancer compound. Eur J Clin Invest 2009; 39: 384-394

[27] Bache M, Bernhardt S, Passin S, Wichmann H, Hein A, Zschornak M, Kappler M,

Taubert H, Paschke R, Vordermark D. Characterization of NVX‐207, a novel betulinic

acid‐derived anti‐cancer compound. Int J Mol Sci 2014; 30:19777-19790

[28] Liebscher G, Vanchangiri K, Mueller T, Feige K, Cavalleri JM, Paschke R. In vitro

anticancer activity of Betulinic acid and derivatives thereof on equine melanoma cell

lines from grey horses and in vivo safety assessment of the compound NVX-207 in two

horses. Chem Biol Interact 2016; 25: 20-29

[29] Gonzalez P, Mader I, Tchoghandjian A, Enzenmüller S, Cristofanon S, Basit F, Debatin

KM, Fulda S. Impairment of lysosomal integrity by B10, a glycosylated derivative

of betulinic acid, leads to lysosomal cell death and converts autophagy into a

detrimental process. Cell Death Differ 2012; 19: 1337-1346

[30] Fischer S, Ronellenfitsch MW, Thiepold AL, Harter PN, Reichert S, Kögel D, Paschke

R, Mittelbronn M, Weller M, Steinbach JP, Fulda S, Bähr O. Hypoxia enhances the

antiglioma cytotoxicity of B10, a glycosylated derivative of betulinic acid. PLoS One

2014; 17: e94921

[31] Fujioka T, Kashiwada Y, Kilkuskie RE, Cosentino LM, Ballas LM, Jiang JB, Janzen

WP, Chen IS, Lee KH. Anti-AIDS agents, 11. Betulinic acid and platanic acid as anti-

HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally

related triterpenoids. J Nat Prod 1994; 57: 243-247

[32] Salzwedel K, Martin DE, Sakalian M. Maturation inhibitors: a new therapeutic class

targets the virus structure. AIDS Rev 2007; 9: 162-172

[33] Martin DE, Blum R, Wilton J, Doto J, Galbraith H, Burgess GL, Smith PC, Ballow C.

Safety and pharmacokinetics of Bevirimat (PA-457), a novel inhibitor of human

immunodeficiency virus maturation, in healthy volunteers. Antimicrob Agents

Chemother 2007; 51: 3063-3066

[34] Smith PF, Ogundele A, Forrest A, Wilton J, Salzwedel K, Doto J, Allaway GP, Martin

DE. Phase I and II study of the safety, virologic effect, and pharmacokinetics/

pharmacodynamics of single-dose 3-o-(3',3'- dimethylsuccinyl)betulinic acid

(bevirimat) against human immunodeficiency virus infection. Antimicrob Agents

Chemother 2007; 51: 3574-3581

[35] Knapp DJ, Harrigan PR, Poon AF, Brumme ZL, Brockman M, Cheung PK. In vitro

selection of clinically relevant bevirimat resistance mutations revealed by "deep"

sequencing of serially passaged, quasispecies-containing recombinant HIV-1. J Clin

Microbiol. 2011; 49: 201-208

[36] Lu W, Salzwedel K, Wang D, Chakravarty S, Freed EO, Wild CT, Li F. A single

polymorphism in HIV-1 subtype C SP1 is sufficient to confer natural resistance to the

maturation inhibitor bevirimat. Antimicrob Agents Chemother 2011; 55: 3324-3329

[37] Regueiro-Ren A, Liu Z, Chen Y, Sin N, Sit SY, Swidorski JJ, Chen J, Venables BL,

Zhu J, Nowicka-Sans B, Protack T, Lin Z, Terry B, Samanta H, Zhang S, Li Z, Beno


-137-

BR, Huang XS, Rahematpura S, Parker DD, Haskell R, Jenkins S, Santone KS, Cockett

MI, Krystal M, Meanwell NA, Hanumegowda U, Dicker IB. Discovery of BMS-

955176, a Second Generation HIV-1 Maturation Inhibitor with Broad Spectrum

Antiviral Activity. ACS Med Chem Lett 2016; 22: 568-572

[38] Morales-Ramirez J, Bogner JR, Molina JM, Lombaard J, Dicker IB, Stock DA,

Degrosky M, Gartland M, Pene Dumitrescu T, Min S, Llamoso C, Joshi SR, Lataillade

M. Safety, efficacy, and dose response of the maturation inhibitor GSK3532795

(formerly known as BMS-955176) plus tenofovir/emtricitabine once daily in treatment-

naive HIV-1-infected adults: Week 24 primary analysis from a randomized Phase IIb

trial. PLoS One 2018; 23: e0205368

[39] Johnson M, Jewell RC, Peppercorn A, Gould E, Xu J, Lou Y, Davies M, Baldwin S,

Tenorio AR, Burke M, Jeffrey J, Johns BA. The safety, tolerability, and

pharmacokinetic profile of GSK2838232, a novel 2nd generation HIV maturation

inhibitor, as assessed in healthy subjects. Pharmacol Res Perspect 2018; 6: e00408

[40] Jäger S, Laszczyk MN, Scheffler A. A preliminary pharmacokinetic study of betulin,

the main pentacyclic triterpene from extract of outer bark of birch (Betulae alba cortex).

Molecules 2008; 13: 3224-3235

[41] Huyke C, Laszczyk M, Scheffler A, Ernst R, Schempp CM. Treatment of actinic

keratoses with birch bark extract: a pilot study. J Dtsch Dermatol Ges 2006; 4: 132-136

[42] Huyke C, Reuter J, Rödig M, Kersten A, Laszczyk M, Scheffler A, Nashan D, Schempp

C. Treatment of actinic keratoses with a novel betulin-based oleogel. A prospective,

randomized, comparative pilot study. J Dtsch Dermatol Ges 2009; 7: 128-133

[43] Scheffler A. Triterpene-containing oleogel-forming agent, triterpene-containing oleogel

and method for producing a triterpene-containing oleogel. European Patent Office,

Patent No. EP 1 758 555 B1, 2009

[44] Scheffler A. Use of an oleogel containing triterpene for healing wounds. United States

Patent Aplication Publication 2012; Patent No. US 2012/0231054 A1

[45] Pflugfelder A, Andonov E, Weide B, Dirschka T, Schempp C, Stockfleth E, Stratigos

A, Krüger-Krasagakis S, Bauer J, Garbe C, Eigentler TK. Lack of activity of betulin-

based Oleogel-S10 in the treatment of actinic keratoses: a randomized, multicentre,

placebo-controlled double-blind phase II trial. Br J Dermatol 2015; 172: 926-932

[46] Haque S, Nawrot DA, Alakurtti S, Ghemtio L, Yli-Kauhaluoma J, Tammela P.

Screening and characterization of antimicrobial properties of semisynthetic betulin

derivatives. Plos One 2014; 9: e102696

[47] Dharmappa KK, Kumar RV, Nataraju A, Mohamed R, Shivaprasad HV, Vishwanath

BS. Anti-inflammatory activity of oleanolic acid by inhibition of secretory

phospholipase A2. Planta Med 2009; 75: 211-215

[48] Barret JP, Podmelle F, Lipový B, Rennekampff HO, Schumann H, Schwieger-Briel A,

Zahn TR, Metelmann HR; BSH-12 and BSG-12 study groups. Accelerated re-

epithelialization of partial-thickness skin wounds by a topical betulin gel: Results of a

randomized phase III clinical trials program. Burns 2017; 43: 1284-1294


-138-

[49] Kindler S, Schuster M, Seebauer C, Rutkowski R, Hauschild A, Podmelle F,

Metelmann C, Metelmann B, Müller-Debus C, Metelmann HR, Metelmann I.

Triterpenes for Well-Balanced Scar Formation in Superficial Wounds. Molecules 2016;

21: E1129

[50] Lipový B, Fiamoli M, Mager R, Jelínková Z, Jarkovský J, Chaloupková Z, Holoubek J,

Suchánek I, Brychta P: Oleogel-s10 to accelerate healing of donor sites: monocentric

results of phase III clinical trial. Acta Chir Plast 2018; 59: 129-134

[51] Frew Q, Rennekampff HO, Dziewulski P, Moiemen N, Moiemen N. BBW-11 study

group, Zahn T, Hartmann B: Betulin wound gel accelerated healing of superficial partial

thickness burns: Results of a randomized, intra-individually controlled, phase III trial

with 12-months follow-up. Burns 2018 Dec 14. pii: S0305-4179(18)30545-X.

doi:10.1016/j.burns.2018.10.019

[52] Ebeling S, Naumann K, Pollok S, Wardecki T, Vidal-Y-Sy S, Nascimento JM, Boerries

M, Schmidt G, Brandner JM, Merfort I. From a traditional medicinal plant to a rational

drug: understanding the clinically proven wound healing efficacy of birch bark extract.

PLoS One 2014; 22: e86147

[53] Woelfle U, Laszczyk MN, Kraus M, Leuner K, Kersten A, Simon-Haarhaus B,

Scheffler A, Martin SF, Müller WE, Nashan D, Schempp CM. Triterpenes promote

keratinocyte differentiation in vitro, ex vivo and in vivo: a role for the transient receptor

potential canonical (subtype) 6. J Invest Dermatol 2010; 130: 113-123.

[54] Schwieger-Briel A, Kiritsi D, Schempp C, Has C, Schumann H: Betulin-Based Oleogel

to Improve Wound Healing in Dystrophic Epidermolysis Bullosa: A Prospective

Controlled Proof-of-Concept Study. Dermatol Res Pract 2017; 2017: 5068969

[55] Scheffler A. The Wound Healing Properties of Betulin from Birch Bark from Bench to

Bedside. Planta Med 2019. doi: 10.1055/a-0850-0224

https://www.ncbi.nlm.nih.gov/pubmed/30559054

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Betulin and its derivatives – precursors of new drugs